Methods for reprograming non-hepatocyte cells into hepatocyte cells

Abstract

A method for inducing reprogramming of a cell of a first type which is not a non-hepatocyte (non-hepatocyte cell), into a cell with functional hepatic drug metabolizing and transporting capabilities, is disclosed. The non-hepatocyte is induced to express or overexpress hepatic fate conversion and maturation factors, cultured in somatic cell culture medium, hepatocyte cell culture medium and hepatocyte maturation medium for a sufficient period of time to convert the non-hepatocyte cell into a cell with hepatocyte-like properties. The iHeps induced according to the methods disclosed herein are functional induced hepatocytes (iHeps) in that they express I and II drug-metabolizing enzymes and phase III drug transporters and show superior drug metabolizing activity compared to iHeps obtained by prior art methods. The iHeps thus provide a cell resource for pharmaceutical applications.

The invention claimed is: 
     
       1. A method for inducing human fibroblast cells into induced hepatocytes-like cells (iHeps), comprising the steps of:
 (a) introducing one or more nucleic acid sequences encoding six factors, wherein the six factors are Hepatocyte nuclear factor 1-alpha (HNF1A), Hepatocyte nuclear factor 4-alpha (HNF4A), Hepatocyte nuclear factor 6-alpha (HNF6), Activating transcription factor 5 (ATF5), Prospero homeobox protein (PROX1), and CCAAT/enhancer-binding protein alpha (CEBPA), into isolated fibroblasts; 
 (b) culturing the cells from the step (a) in a somatic cell medium; 
 (c) expanding the cells from the step (b) in a hepatocyte cell culture medium; and 
 (d) culturing the cells from the step (c) in a hepatocyte maturation medium wherein expression of the six factors is effective to induce formation of iHeps, wherein the iHeps do not express α-fetoprotein, and wherein the iHeps secret albumin, and are capable of taking up LDL, incorporating indocyanine green (ICG), synthesizing and storing glycogen and accumulating fat. 
 
     
     
       2. The method of  claim 1 , wherein the step (a) further comprises treating the cells to upregulate MYC and downregulate p53. 
     
     
       3. The method of  claim 2 , wherein the step (a) comprises transfecting the cells with a vector expressing p53 siRNA and inhibiting the expression of p53 siRNA at the end of the step (c). 
     
     
       4. The method of  claim 1 , wherein in the step (a) the cells are transformed with nucleic acids as set forth by SEQ ID NOs: 1-7, respectively. 
     
     
       5. The method of  claim 1 , wherein in the step (b) the cells are cultured in the somatic cell culture medium for a period of at least 7 days. 
     
     
       6. The method of  claim 1  wherein in the step (c) the cells are cultured in the hepatocyte cell culture medium for a period of about 15 to 30 days, preferably, 18-30 days, more preferably about 18 days. 
     
     
       7. The method of  claim 1  wherein in the step (d) the cells are cultured in the hepatocyte maturation medium for a period of at least 5 days. 
     
     
       8. The method of  claim 1 , wherein the fibroblast cells are derived from a mammal, the method further comprising identifying iHeps by detecting the expression of at least one hepatic marker selected from the group consisting of albumin, Cytochrome P450 (CYP)3A4 and CYPB6, glycogen synthesis and storage, and/or fatty droplet accumulation. 
     
     
       9. The method of  claim 8 , wherein the mammal is selected from the group consisting of human, rat, mouse, monkey, dog, cat, cattle, rabbit, horse and pig. 
     
     
       10. The method of  claim 1 , wherein the iHeps express at least one drug metabolizing enzyme selected from the group consisting of CYP3A4, CYPB6, CYP1A2, CYP2C9, CYP2C19, or combinations thereof and optionally, wherein MYC expression level in the iHeps is lower than the MYC expression level found in hepatocytes obtained from the corresponding organism. 
     
     
       11. The method of  claim 1 , wherein the non-hepatocyte cells are fibroblast cells, and optionally wherein the iHeps express E-cadherin, the iHeps do not express one or more of the fibroblast marker genes selected from COL1A1, PDGFRB, THY1 and α-fetoprotein, or a combination thereof. 
     
     
       12. The method of  claim 1 , wherein the iHeps at least one drug metabolic phase II enzyme or phase II transporter selected from the group consisting of CYP1A2, CYP2C9, CYP2C19, UDP glucuronosyltransferase (UGT)1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, GSTA1, UGT2B7, UGT2515, Microsomal glutathione-S-transferase 1 (MGST1), nicotinamide N-methyltransferase (NNMT), NTCP, organic anion-transporting polypeptide 1B3 (OATP1B3), Multidrug resistance protein(MRP)6, MRP2, Flavin-containing monooxygenase 5 (FMO5), Monoamine oxidase (MAO)A, MAOB, and epoxide hydrolase 1 (EPHX1). 
     
     
       13. The method of  claim 1 , wherein the metabolic activity of at least one of CYP3A4, CYPB6, CYP1A2, CYP2C9, and CYP2C19 is at least 50% higher than the activity of the same enzyme in ES-Heps (embryonic stem cell (ESC)-derived hepatocytes) obtained from the same organism. 
     
     
       14. The method of  claim 13 , wherein the metabolic activity of at least one of CYP3A4, CYPB6, CYP1A2, CYP2C9, and CYP2C19 is 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, higher than the activity in ES-Heps. 
     
     
       15. The method of  claim 13 , wherein the metabolic activity of at least one of CYP3A4, CYPB6, CYP1A2, CYP2C9, and CYP2C19 is at least 100-fold higher than that of ES-Heps. 
     
     
       16. A method for inducing human fibroblast cells into induced hepatocytes-like cells (iHeps), comprising the steps of:
 (a) introducing one or more nucleic acid sequences encoding HNF1A, HNF4A, HNF6, ATF5, PROX1, and CEBPA, into isolated human fibroblasts; 
 (b) culturing the cells from the step (a) in a somatic cell medium; 
 (c) expanding the cells from the step (b) in a hepatocyte cell culture medium; and 
 (d) culturing the cells from the step (c) in a hepatocyte maturation medium such that iHeps form, wherein the iHeps do not express α-fetoprotein, and wherein the iHeps secret albumin, and are capable of taking up LDL, incorporating indocyanine green (ICG), synthesizing and storing glycogen and accumulating fat; 
 the method further comprising identifying iHeps by detecting the expression of at least one hepatic marker selected from the group consisting of albumin, Cytochrome P450 (CYP)3A4 and CYPB6, glycogen synthesis and storage, and/or fat accumulation, 
 wherein the mammal is a human. 
 
     
     
       17. A method for inducing human fibroblast cells into induced hepatocytes-like cells (iHeps), comprising the steps of:
 (a) introducing one or more nucleic acid sequences encoding HNF1A, HNF4A, HNF6, ATF5, PROX1, and CEBPA, into isolated human fibroblasts; 
 (b) culturing the cells from the step (a) in a somatic cell medium; 
 (c) expanding the cells from the step (b) in a hepatocyte cell culture medium; and 
 (d) culturing the cells from the step (c) in a hepatocyte maturation medium such that iHeps form, wherein the iHeps do not express α-fetoprotein, and wherein the iHeps secret albumin, and are capable of taking up LDL, incorporating indocyanine green (ICG), synthesizing and storing glycogen and accumulating fat.